Promoter Escape with Bacterial Two-component σ Factor Suggests Retention of σ Region Two in the Elongation Complex

Sengupta, Shreya; Prajapati, Ranjit Kumar; Mukhopadhyay, Jayanta

doi:10.1074/jbc.m115.666008

Cited by 8 publications

(6 citation statements)

References 27 publications

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“…The rpoD gene encodes the σ 70 subunit of RNA polymerase, which is the principal σ factor in the cell. RNA polymerase holoenzyme containing σ 70 is the DNA‐binding species at σ 70 ‐dependent promoters and σ 70 is classically thought to dissociate from RNA polymerase following promoter escape (Hansen & McClure, 1980; Nickels et al, 2005), with some noted exceptions (Daube & von Hippel, 1999; Ko et al, 1998; Sengupta et al, 2015; Wade & Struhl, 2004). Thus, the location of σ 70 (RpoD) along the DNA has been used as a surrogate to indicate the location of σ 70 ‐dependent promoters along the genomes of various bacterial genera (Kroger et al, 2012; Mooney et al, 2009; Ramsey et al, 2015; Singh & Grainger, 2013; Singh et al, 2014).…”

Section: Resultsmentioning

confidence: 99%

H‐NS‐like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription

Lippa

Gebhardt

Dove

2020

Molecular Microbiology

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The H‐NS‐like proteins MvaT and MvaU act coordinately as global repressors in Pseudomonas aeruginosa by binding to AT‐rich regions of the chromosome. Although cells can tolerate loss of either protein, identifying their combined regulatory effects has been challenging because the loss of both proteins is lethal due to induction of prophage Pf4 and subsequent superinfection of the cell. In other bacteria, H‐NS promotes the cellular fitness by inhibiting intragenic transcription from AT‐rich target regions, preventing them from sequestering RNA polymerase; however, it is not known whether MvaT and MvaU function similarly. Here, we utilize a parental strain that cannot be infected by Pf4 phage to define the collective MvaT and MvaU regulon and demonstrate that the combined loss of both MvaT and MvaU leads to increased intragenic transcription from loci directly controlled by these proteins. We further show that the loss of MvaT and MvaU leads to a striking redistribution of RNA polymerase containing σ70 to genomic regions vacated by these proteins. Our findings suggest that the ability of H‐NS‐like proteins to repress intragenic transcription is a universal function of these proteins and point to a second mechanism by which MvaT and MvaU may contribute to the growth of P. aeruginosa.

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Section: Resultsmentioning

confidence: 99%

H‐NS‐like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription

Lippa

Gebhardt

Dove

2020

Molecular Microbiology

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“…To verify whether the purified RNAPs were transcriptionally active, an in vitro transcription assay was performed with 100 nM of each RNAP and an exogenously added phage promoter P tetR (10 mM). The transcripts were labelled with αP 32 labelled radio nucleotides using the protocol as in [30]. The transcripts were run in a 12% Urea PAGE and scanned in a Storage Phosphoimager (Typhoon trio +GE Healthcare) (Fig.…”

Section: Methodsmentioning

confidence: 99%

Mycobacteriophage D29 induced association of Mycobacterial RNA polymerase with ancillary factors leads to increased transcriptional activity

et al. 2022

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Mycobacteriophage D29 infects species belonging to the genus Mycobacterium including the deadly pathogen Mycobacterium tuberculosis. D29 is a lytic phage, although, related to the lysogenic mycobacteriophage L5. This phage is unable to lysogenize in mycobacteria as it lacks the gene encoding the phage repressor. Infection by many mycobacteriophages cause various changes in the host that ultimately leads to inactivation of the latter. One of the host targets often modified in the process is RNA polymerase. During our investigations with phage D29 infected Mycobacterium smegmatis (Msm) we observed that the promoters from both phage, and to a lesser extent those of the host were found to be more active in cells that were exposed to D29, as compared to the unexposed. Further experiments indicate that the RNA polymerase purified from phage infected cells possessed higher affinity for promoters particularly those that were phage derived. Comparison of the purified RNA polymerase preparations from infected and uninfected cells showed that several ancillary transcription factors, Sigma factor F, Sigma factor H, CarD and RbpA are prominently associated with the RNA polymerase from infected cells. Based on our observations we conclude that the higher activity of RNA polymerase observed in D29 infected cells is due to its increased association with ancillary transcription factors.

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“…(1) Displacement of σR3.2, which otherwise impedes the nascent RNA 5′‐end; removal of σR3.2 together with σR4 allows nascent RNA to enter the RNA exit channel. (2) Weakening of RNAP interactions with σ, mainly through destabilization of the σR4–β interaction . (3) Breaking σ70 interactions with the promoter (σR2.3–R2.4 with the −10 element, σR4.2 with −35 element).…”

Section: Rnap Rpo Formation and Tss Selectionmentioning

confidence: 99%

Studying transcription initiation by RNA polymerase with diffusion‐based single‐molecule fluorescence

Alhadid

Chung

Lerner³

et al. 2017

Protein Science

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Over the past decade, fluorescence-based single-molecule studies significantly contributed to characterizing the mechanism of RNA polymerase at different steps in transcription, especially in transcription initiation. Transcription by bacterial DNA-dependent RNA polymerase is a multistep process that uses genomic DNA to synthesize complementary RNA molecules. Transcription initiation is a highly regulated step in E. coli, but it has been challenging to study its mechanism because of its stochasticity and complexity. In this review, we describe how single-molecule approaches have contributed to our understanding of transcription and have uncovered mechanistic details that were not observed in conventional assays because of ensemble averaging.

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Promoter Escape with Bacterial Two-component σ Factor Suggests Retention of σ Region Two in the Elongation Complex

Cited by 8 publications

References 27 publications

H‐NS‐like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription

H‐NS‐like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription

Mycobacteriophage D29 induced association of Mycobacterial RNA polymerase with ancillary factors leads to increased transcriptional activity

Studying transcription initiation by RNA polymerase with diffusion‐based single‐molecule fluorescence

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